Methods for treating bacterial infection

ABSTRACT

The present disclosure relates to molecules that function as selective modulators (i.e., inhibitors and agonists) of the Ras-homologous (Rho) family of small GTPases and, in particular, CDC42 GTPase, and their use to treat bacterial infection including systemic infection from sources such as  Staphylococcus aureus  and  Streptococcus pyogenes.

CROSS REFERENCE TO RELATED APPLICATION

The present invention claims the benefit of priority as acontinuation-in-part to U.S. Non-Provisional patent application Ser. No.14/561,787, filed Dec. 5, 2014, which claims the benefit of priority toU.S. Provisional Patent Application Ser. No. 61/912,723, filed Dec. 6,2013, and to U.S. Non-Provisional patent application Ser. No.13/773,871, filed Feb. 22, 2013, which claims the benefit of priority toU.S. Provisional Patent Application Ser. No. 61/671,054, filed Jul. 12,2012, Provisional Patent Application Ser. No. 61/644,798, filed May 9,2012, and U.S. Provisional Patent Application Ser. No. 61/601,807, filedFeb. 22, 2012; all of which are incorporated herein by reference.

GOVERNMENT LICENSE RIGHTS

This invention was made with government support under Grant No. 1 R15HL092504-01 awarded by the National Institutes of Health, NationalHeart, Lung and Blood Institute. The U.S. Government has certain rightsin the invention disclosed herein.

FIELD

The present disclosure relates to molecules that function as selectivemodulators (i.e., inhibitors and agonists) of the Ras-homologous (Rho)family of small GTPases and, in particular, CDC42 GTPase, and their useto treat bacterial infection including systemic infection from sourcessuch as Staphylococcus aureus and Streptococcus pyogenes.

BACKGROUND

In the US, S. aureus is the most common etiologic agent in systemicinfection and in biofilm-mediated infection of implanted devices.Treatment is complicated by the steady emergence of antibioticresistance and by increases in elderly, immunocompromised populations,prevalence in the use of surgically implanted devices, and by theability of both resistant and susceptible strains to persistasymptomatically months to years after the withdrawal of antimicrobialtherapy. Severe infection is associated with high rates of mortality(11%-43%) and with chronic, debilitating morbidities that includeinfective endocarditis, osteomyelitis, and recurrent lung infection. Thecurrent cost-of-care is estimated at $10 billion annually for treatmentof infection by methicillin resistant S. aureus (MRSA) alone.

S. pyogenes is the cause of many human diseases, ranging from mild skininfections to life-threatening systemic diseases. An estimated 700million infections occur worldwide each year, and over 650,000 cases ofsevere, invasive infections that have a mortality rate of approximately25%.

Strategies for improving treatment options for S. aureus and S. pyogeneshave included the creation of new antibiotics and the development ofadjunctive therapeutics.

SUMMARY

Compounds have been examined that inhibit host cell invasion through amevalonate independent mechanism. It was found that ML 141 inhibitsendothelial cell invasion and intracellular persistence (see FIGS. 1, 2Aand 2B). The mode-of-action of ML 141 is distinct from that of othertherapeutics used in treating invasions, such as some statins. ML 141inhibits GTP-binding at the activation site of CDC42. Some statinscompete for substrate binding within the catalytic site of3-hydroxy-3-methylglutaryl (HMG)-CoA reductase. Similar to ML 141, somestatins functionally inactivate CDC42 through an indirect mechanism. ML141 binds directly to CDC42 with a high degree of specificity for thissmall-GTPase. The mechanism of inhibition by ML 141 also includesdecreasing host cell adhesion to fibronectin, the extracellular matrixprotein used by S. aureus for gaining host cell entry. Inhibition bysome statins is not specific for CDC42, but rather is due to diminishedlevels of isoprenoid intermediates formed from mevalonate within thecholesterol biosynthesis pathway. The isoprenoid intermediates farnesylpyrophosphate and geranylgeranyl pyrophosphate provide membraneanchoring and protein-protein interactions for CaaX-motif containingproteins that include CDC42. In this way, ML 141 and some statinsfunctionally inhibit CDC42 through different mechanisms.

Host CDC42 may provide a central target in the treatment of invasiveinfection. CDC42 is used by S. aureus to facilitate uptake into hostcells and is targeted by staphylococcal toxins for tunneling throughendothelial cells to the underlying matrix. This selective use of CDC42is consistent with invasion by Streptococcus pneumonia, the etiologicagent of community-acquired pneumonia, in that redundancy amongstsmall-GTPases fails to restore invasiveness in cells expressingdominant-negative CDC42. The use of CDC42 to gain host cell entryextends beyond bacterial pathogens to viral infection, facilitating bothuptake and replication.

Until recently, the biological relevance of invasiveness by S. aureushad been challenged. S. aureus had been considered an extracellularpathogen, and intracellular residency appeared to be an in vitroartifact. However, in vivo and clinical evidence supports the conceptthat intracellular residency by S. aureus contributes significantly topathogenesis by stimulating pro-inflammatory and pro-coagulantresponses, by enabling evasion of antibiotics and immune cells, and byestablishing intracellular bacterial reservoirs as sources of chronicinfection. Numerous questions exist regarding the biological consequenceof host cell invasion by S. aureus, including whether uptake bynon-immune cells serves solely as a mechanism of evasion, or whetherthis uptake serves as a mechanism of host defense. Studies using ML 141investigate whether the direct inhibition of CDC42 impedes the roles ofthis protein in innate immunity.

ML 141 remains largely uncharacterized with respect to antibacterialactivity. Synthesis initially was described within a series of compoundspredicted to possess antibacterial activity. However, antimicrobial datawere not presented. Research found that the compound inhibitsGTP-loading of CDC42 with a high degree of specificity for thissmall-GTPase.

Embodiments disclosed herein provide a method of suppressing microbialinfection comprising administering ML 141 or its analogs to cells, wheresuch analogs include the following structure:

Where Ar′ is methoxyphenyl (-Ph-O-Me), R′ is hydrogen, and R and Ar arephenylmethylene (-Ph-(2-CH₂)—), wherein the phenyl is attached to the Arposition and the methylene is attached to the R position; or Ar′ ismethoxyphenyl (-Ph-O-Me), R′ is methyl, R is hydrogen, and Ar is phenyl;or Ar′ is halophenyl (-Ph-halogen), R′ is hydrogen, R is hydrogen, andAr is phenylpolyethylene glycol methyl ether (-Ph-(O—CH₂—CH₂)_(n)—O-Me)wherein n is any integer.

In some embodiments, the microbial infection is from Staphylococcusaureus and/or Streptococcus pyogenes and/or an intracellular pathogenthat uses fibronectin binding to gain entry into a host cell. In furtherembodiments, the administering step includes providing ML 141 or itsanalogs adjacent to the cells. In certain embodiments, the administeringstep includes testing ML 141 on the cells. In some embodiments, theadministering step includes testing at least one analog on the cells. Infurther embodiments, suppressing microbial infection includessuppressing initial microbial infection. In certain embodiments,suppressing microbial infection includes suppressing persistentmicrobial infection. In some embodiments, the administering stepincludes providing approximately 1 μM of ML 141 or its analogs. In evenfurther embodiments, the administering step includes providingapproximately 10 μM of ML 141 or its analogs. In certain embodiments,the administering step occurs subsequent to the onset of microbialinfection. In some embodiments, the cells are animal cells, are humancells, are from a human and/or are from an animal. In furtherembodiments, the cells express CDC42.

Embodiments disclosed herein provide a method of suppressing microbialinfection comprising providing a chemical including the followingstructure:

where Ar′ is methoxyphenyl (-Ph-O-Me), R′ is hydrogen, and R and Ar arephenylmethylene (-Ph-(2-CH₂)—), wherein the phenyl is attached to the Arposition and the methylene is attached to the R position; or Ar′ ismethoxyphenyl (-Ph-O-Me), R′ is methyl, R is hydrogen, and Ar is phenyl;or Ar′ is halophenyl (-Ph-halogen), R′ is hydrogen, R is hydrogen, andAr is phenylpolyethylene glycol methyl ether (-Ph-(O—CH₂—CH₂)_(n)—O-Me)wherein n is any integer; and providing the chemical to cells. In someembodiments, the method further comprises providing a pharmaceuticallyaccepted solvent or delivery vehicle selected from the group consistingof polyethylene glycol (PEG), dimethyl sulfoxide, ethanol, andcombinations thereof. In certain embodiments, the solvent or deliveryvehicle is polyethylene glycol. In other embodiments, the halophenyl ischlorophenyl.

Embodiments disclosed herein provide a method of suppressing microbialinfection comprising administering ML 141 or its analogs to cells, wheresuch analogs include the following structures:

where Ar is phenyl, acetamidophenyl (-Ph-NH—CO-Me), or propanamidophenyl(Ph-NH—CO-Et), R is hydrogen, R′ is hydrogen, and Ar′ is methoxyphenyl(-Ph-O-Me), acetamidophenyl (-Ph-NH—CO-Me), or dimethylaminophenyl(Ph-N-Me2); and

where Ar is phenyl and Ar′ is methoxyphenyl (-Ph-O-Me).

BRIEF DESCRIPTION OF THE DRAWINGS

The above-mentioned and other features of this disclosure, and themanner of attaining them, will become more apparent and the disclosureitself will be better understood by reference to the followingdescription of embodiments of the disclosure taken in conjunction withthe accompanying drawings, wherein:

FIG. 1 illustrates ML 141 limits host cell invasion. The monocytic cellline U937 was pretreated with the vehicle control dimethyl sulfoxide(DMSO) or with ML 141 (0.05 μM, 1 h) followed by infection with AlexaFluor 488-labeled S. aureus (1 hour). The % of 488⁺ cells decreased withML 141 treatment.

FIG. 2A is a schematic model of Staphylococcus aureus host cell invasionin the absence of ML 141. Step 1: In the absence of ML 141, S. aureusbound to host fibronectin interacts with the host cell integrin α₅β₁,stimulating GTP-loading and activation of CDC42. Step 2: GTP-loading ofCDC42 increases affinity for the p85α regulatory subunit ofphosphoinositide 3-kinase (PI3K). CDC42, coupled to PI3K through thep85α subunit, positions the catalytic domain p110α in proximity withphosphoinositide 4,5-bisphosphate (PI_(4,5)). Step 3: The product of thephosphorylation of PI_(4,5) by p110α is PI 3,4,5-trisphosphate (PIP₃),capable of promoting endocytosis of the bacterium/fibronectin/integrincomplex, as illustrated by Step 4.

FIG. 2B is a schematic model of Staphylococcus aureus host cellinhibition by ML 141. Step 1: In response to the inhibition ofGTP-loading of CDC42 by ML 141, CDC42 remains uncoupled from p85α.Consequently, p110α remains within the cytosol. By sequestering p110αwithin the cytosol, membrane-bound PI_(4,5) is not accessible,diminishing PIP₃ production. Step 2: In the absence of PIP₃, endocyticuptake of the bacterium/fibronectin/integrin complex is limited,protecting the host cell from bacterial invasion (models are based onreferences indicated in text and on current study).

FIG. 3 illustrates how invasion stimulates coupling between CDC42 andthe downstream effector phosphoinositide 3-kinase (PI3K) p85. Host cells(human embryonic kidney cells) were infected for 15 or 60 min, lysatesimmunoprecipitated (IP) with anti-CDC42, and immunoblot (IB) probed withanti-p85 followed by Alexa Fluor anti-rabbit 800CW. Fluorescence wasdetected using the Odyssey Infrared Imaging System. Integratedintensities and background correction were performed using Odysseysoftware.

FIG. 4A-1 illustrates by histogram overlay how ML 141 inhibits host cellinvasion. Human umbilical vein endothelial cells (HUVEC) were pretreated(18 hours) with the vehicle control polyethylene glycol (PEG) or ML 141(10 μM) suspended in PEG. Following infection by Alexa Fluor 488-labeledStaphylococcus aureus (1 hour), extracellular bacteria were removedusing the antimicrobials gentamicin and lysostaphin. Infected cells(identified by 488 fluorescence) were detected by flow cytometry andrepresented by histogram overlay.

FIG. 4A-2 illustrates by averaged mean fluorescence intensity values howML 141 inhibits host cell invasion. Human umbilical vein endothelialcells (HUVEC) were pretreated (18 hours) with the vehicle controlpolyethylene glycol (PEG) or ML 141 (10 μM) suspended in PEG. Followinginfection by Alexa Fluor 488-labeled Staphylococcus aureus (1 hour),extracellular bacteria were removed using the antimicrobials gentamicinand lysostaphin. Infected cells (identified by 488 fluorescence) weredetected by flow cytometry and represented by histogram overlay asillustrated in FIG. 4A-1 and averaged mean fluorescence intensity values(*less than vehicle control; p s 0.001 by Student's t-test;n=3-5/treatment).

FIG. 4B-1 illustrates by histogram overlay how ML 141 inhibits host cellinvasion. Human umbilical vein endothelial cells (HUVEC) were pretreated(18 hours) with the vehicle control dimethyl sulfoxide (DMSO) or ML 141(10 μM) suspended in dimethyl sulfoxide (DMSO). Following infection byAlexa Fluor 488-labeled Staphylococcus aureus (1 hour), extracellularbacteria were removed using the antimicrobials gentamicin andlysostaphin. Infected cells (identified by 488 fluorescence) weredetected by flow cytometry and represented by histogram overlay.

FIG. 4B-2 illustrates by averaged mean fluorescence intensity values howML 141 inhibits host cell invasion. Human umbilical vein endothelialcells (HUVEC) were pretreated (18 hours) with the vehicle controldimethyl sulfoxide (DMSO) or ML 141 (10 μM) suspended in dimethylsulfoxide (DMSO). Following infection by Alexa Fluor 488-labeledStaphylococcus aureus (1 hour), extracellular bacteria were removedusing the antimicrobials gentamicin and lysostaphin. Infected cells(identified by 488 fluorescence) were detected by flow cytometry andrepresented by histogram overlay as illustrated in FIG. 4B-1 andaveraged mean fluorescence intensity values (*less than vehicle control;p≦0.001 by Student's t-test; n=3-5/treatment).

FIG. 5 is a graph illustrating ML 141 inhibiting invasion with shorterduration exposure under serum starved conditions. RAW 264.7 cells wereserum-starved overnight then pretreated for 1 hour with the vehiclecontrol dimethyl sulfoxide (DMSO) or with ML 141 (10 μM). Followinginfection with Staphylococcus aureus (1 hour), extracellular bacteriawere eliminated using the antimicrobials gentamicin and lysostaphin (45minutes), and intracellular bacteria recovered by permeabilizing thehost cells. Serial dilutions of the recovered intracellular bacteriawere incubated on tryptic soy agar, colonies enumerated, and colonyforming units (CFU)/ml calculated (*less than vehicle control; p≦0.05 byStudent's t-test; n=3/treatment).

FIG. 6A illustrates that neither cytotoxicity nor bactericidal activityis detected. Human umbilical vein endothelial cells (HUVEC) werepretreated (18 hours) with the vehicle control polyethylene glycol (PEG)or with ML 141 and viability assessed using an XTT assay. The formazandye produced in living cells was detectable at an absorbance wavelengthof 490 nanometers and used as an indicator of cell viability. Absorbancevalues were not different amongst the groups (p>0.05 by one-way ANOVA;n=3/treatment).

FIG. 6B illustrates that neither cytotoxicity nor bactericidal activityis detected. Cultures of Staphylococcus aureus that had been incubatedwith the vehicle control DMSO or with ML 141 (1 hour, 37° C., 225 rpm)were serially diluted, plated onto blood agar, and incubated (18 hours,37° C.). Colonies were enumerated and colony forming units (CFU)/mldetermined. CFU/ml were not different amongst the groups (p>0.05 byt-test; n=2/treatment).

FIG. 7 is a graph illustrating suppression of intracellular populationsustained over time. Human umbilical vein endothelial cells (HUVEC) werepretreated (1.0 μM, 18 hours) with the vehicle control dimethylsulfoxide (DMSO) or ML 141 and infected with Staphylococcus aureus (1hour). At 48 hours, intracellular bacteria were recovered, serialdilutions incubated on tryptic soy agar and colonies enumerated. Dataare presented as % control, ±SEM (*less than vehicle control; p≦0.05 byt-test; n=3-5/treatment).

FIG. 8A is an image depicting ML 141 limiting actin stress fiberdepolymerization during infection. Human umbilical vein endothelialcells (HUVEC) were incubated with ML 141 or with the vehicle controlpolyethylene glycol (PEG) 18-20 h prior to infection with Staphylococcusaureus (1 h). Actin was detected using Alexa Fluor 488 phalloidin.Arrows indicate intact actin stress fibers. Arrowhead indicates celllacking actin stress fibers. 100 cells/treatment were evaluated fromrandomly selected fields. Data are presented as the % of cells in whichactin stress fibers were not detected. Scale bar is 50 μm.

FIG. 8B is an image depicting RSM 06 limiting actin stress fiberdepolymerization during infection. Human umbilical vein endothelialcells (HUVEC) were incubated with RSM 06 or with the vehicle controlpolyethylene glycol (PEG) 18-20 h prior to infection with Staphylococcusaureus (1 h). Actin was detected using Alexa Fluor 488 phalloidin. 200cells/treatment were evaluated from randomly selected fields. Data arepresented as the % of cells in which actin stress fibers were notdetected. Scale bar is 50 μm.

FIG. 8C is an image depicting RSM 16 limiting actin stress fiberdepolymerization during infection. Human umbilical vein endothelialcells (HUVEC) were incubated with RSM 16 or with the vehicle controlpolyethylene glycol (PEG) 18-20 h prior to infection with Staphylococcusaureus (1 h). Actin was detected using Alexa Fluor 488 phalloidin. 200cells/treatment were evaluated from randomly selected fields. Data arepresented as the % of cells in which actin stress fibers were notdetected. Scale bar is 50 μm.

FIG. 9 is an image depicting ML 141 limiting damage to host cells. Humanumbilical vein endothelial cells were incubated with the vehicle controlpolyethylene glycol (PEG) or with ML 141 (10 μM) 18-20 h prior toinfection with Staphylococcus aureus at a multiplicity of infection of30 (2 h). Following infection, extracellular bacteria were removed byincubating with lysostaphin and gentamicin (45 min), antimicrobials withlimited mammalian membrane permeability. Antimicrobial-containing mediawas replenished and incubation extended an additional 24 h. Cells werefixed, stained, and examined by transmission electron microscopy. Whitearrow indicates mitochondria. Black arrow indicates filopodia. Whitearrowhead indicates membranous structure surrounding bacteria. Blackarrowhead indicates dividing bacteria. Scale bar is 5 μm.

FIG. 10A is an image depicting ML 141 inhibition of CDC42 limiting theformation of adhesion complexes. Human umbilical vein endothelial cells(HUVEC) were incubated (18-20 h) with the vehicle control dimethylsulfoxide (DMSO) or with ML 141 (10 μM). Pretreated HUVEC were fixed,permeabilized, blocked, and stained with anti-vinculin followed byanti-mouse Alexa Fluor 488. Arrows indicate vinculin-containingcomplexes. Data are presented as the % of cells wherevinculin-containing adhesion complexes were detected. 100-200cells/treatment were evaluated from randomly selected fields (p<0.001 byχ²). Scale bar 50 μm.

FIG. 10B is a graph illustrating that expression of the 131 integrinsubunit remains substantially unchanged in HUVEC incubated with DMSO orML 141 (10 μM). Pretreated HUVEC were stained with PE conjugated anti-β1and examined using flow cytometry (no difference between groups wasdetected, t-test, p>0.05).

FIG. 10C is a graph illustrating that ML 141 decreases cell adhesion tofibronectin-coated surface. HUVEC were pretreated as in FIGS. 10A and10B, lifted from culture dishes using cell scrapers, and re-plated (2 h)onto non-tissue culture treated plates coated with fibronectin andblocked with bovine serum albumin. Following extensive washes, cellswere recovered using trypsin and counted using an Accuri C6 flowcytometer (*p<0.05 by t-test on log-transformed data pooled from 2independent experiments, n=8/treatment).

FIG. 11 is a graph illustrating suppression of intracellular populationsustained over time. Human umbilical vein endothelial cells (HUVEC) werepretreated (10 μM, 18 hours) with the vehicle control dimethyl sulfoxide(DMSO) or ML 141 and infected with Staphylococcus aureus (1 hour). At 48hours, intracellular bacteria were recovered, serial dilutions incubatedon tryptic soy agar and colonies enumerated. Data are presented as %control, ±SEM (*less than vehicle control; p≦0.05 by t-test;n=3-5/treatment).

FIG. 12 is a graph illustrating that ML 141 inhibition of cell invasionby S. pyogenes is reversible. HUVEC were pretreated with ML 141 (10 μM)or with dimethyl sulfoxide (DMSO) as the vehicle control (1 h).Following pretreatment, ML 141-containing media was removed from thewashout samples and replaced with DMSO-containing media for 60 min priorto incubation with Streptococcus pyogenes at a multiplicity of infectionof 30 (2 h). Extracellular bacteria were removed by gentamicin, anantimicrobial with limited mammalian membrane permeability.Intracellular bacteria were released from HUVEC into the medium byincubation in cold water (5 min). Serial dilutions were incubated (16 h)on Todd Hewitt broth/blood agar plates and colonies enumerated todetermine colony forming units (CFU)/ml (*P<0.05 by one-way ANOVAfollowed by Student-Newman-Keuls post-hoc analysis; n=4/treatment).

FIG. 13 is an image depicting ML 141 decreasing actin stress fiberdepolymerization during infection. HUVEC were incubated with vehiclecontrol dimethyl sulfoxide (DMSO) or with ML 141 (10 μM) 18-20 h priorto infection with Streptococcus pyogenes at a multiplicity of infectionof 30 (2 h). Actin was detected using Alexa Fluor 488 phalloidin. Dataare presented as the percentage of HUVEC with no detectable actin stressfibers. 100-200 cells/treatment were evaluated from randomly selectedfields. Scale bar is 50 μm (P<0.05 by χ² test of association).

FIG. 14A is a depiction of the chemical structure of the RSM 04structural analog of ML 141 and a chart indicating its ability toinhibit Staphylococcus aureus invasion of human umbilical veinendothelial cells as compared to ML141 and a PEG control.

FIG. 14B is a depiction of the chemical structure of the RSM 05structural analog of ML 141 and a chart indicating its ability toinhibit Staphylococcus aureus invasion of human umbilical veinendothelial cells as compared to ML141 and a PEG control.

FIG. 14C is a depiction of the chemical structure of the RSM 06structural analog of ML 141 and a chart indicating its ability toinhibit Staphylococcus aureus invasion of human umbilical veinendothelial cells as compared to ML141 and a PEG control.

FIG. 14D is a depiction of the chemical structure of the RSM 07structural analog of ML 141 and a chart indicating its ability toinhibit Staphylococcus aureus invasion of human umbilical veinendothelial cells as compared to ML141 and a PEG control.

FIG. 14E is a depiction of the chemical structure of the RSM 11structural analog of ML 141 and a chart indicating its ability toinhibit Staphylococcus aureus invasion of human umbilical veinendothelial cells as compared to ML141 and a PEG control.

FIG. 14F is a depiction of the chemical structure of the RSM 12structural analog of ML 141 and a chart indicating its ability toinhibit Staphylococcus aureus invasion of human umbilical veinendothelial cells as compared to ML141 and a PEG control.

FIG. 14G is a depiction of the chemical structure of the RSM 13structural analog of ML 141 and a chart indicating its ability toinhibit Staphylococcus aureus invasion of human umbilical veinendothelial cells as compared to ML141 and a PEG control.

FIG. 14H is a depiction of the chemical structure of the RSM 14structural analog of ML 141 and a chart indicating its ability toinhibit Staphylococcus aureus invasion of human umbilical veinendothelial cells as compared to ML141 and a PEG control.

FIG. 14I is a depiction of the chemical structure of the RSM 15structural analog of ML 141 and a chart indicating its ability toinhibit Staphylococcus aureus invasion of human umbilical veinendothelial cells as compared to ML141 and a PEG control.

FIG. 14J is a depiction of the chemical structure of the RSM 16structural analog of ML 141 and a chart indicating its ability toinhibit Staphylococcus aureus invasion of human umbilical veinendothelial cells as compared to ML141 and a PEG control.

FIG. 14K is a depiction of the chemical structure of the RSM 17structural analog of ML 141 and a chart indicating its ability toinhibit Staphylococcus aureus invasion of human umbilical veinendothelial cells as compared to ML141 and a PEG control.

FIG. 14L is a depiction of the chemical structure of the RSM 18structural analog of ML 141 and a chart indicating its ability toinhibit Staphylococcus aureus invasion of human umbilical veinendothelial cells as compared to ML141 and a PEG control.

FIG. 14M is a depiction of the chemical structure of the RSM 19structural analog of ML 141 and a chart indicating its ability toinhibit Staphylococcus aureus invasion of human umbilical veinendothelial cells as compared to ML141 and a PEG control.

FIG. 15A is a depiction of the chemical structure of the RSM 27structural analog of ML 141.

FIG. 15B is a depiction of the chemical structure of the RSM 28structural analog of ML 141.

FIG. 15C is a depiction of the chemical structure of the RSM 29structural analog of ML 141.

FIG. 15D is a depiction of the chemical structure of the RSM 30structural analog of ML 141.

FIG. 15E is a depiction of the chemical structure of the RSM 31structural analog of ML 141.

FIG. 15F is a depiction of the chemical structure of the RSM 32structural analog of ML 141.

FIG. 15G is a depiction of the chemical structure of the RSM 33structural analog of ML 141.

Corresponding reference characters indicate corresponding partsthroughout the several views. Although the drawings representembodiments of the present disclosure, the drawings are not necessarilyto scale and certain features may be exaggerated in order to betterillustrate and explain the present disclosure.

DETAILED DESCRIPTION OF THE EXEMPLARY EMBODIMENTS

The embodiments disclosed below are not intended to be exhaustive orlimit the disclosure to the precise forms disclosed in the followingdetailed description. Rather, the embodiments are chosen and describedso that others skilled in the art may utilize their teachings.

Innovation

The ML 141 compound has been used to address whether targeting CDC42limits persisting intracellular populations, diminishing this source ofchronic infection and initiates pleiotropic effects by interrupting thePI3K signaling pathway.

Findings will impact the characterization of this potentiallyfirst-in-class molecule for the development of adjunctive therapeutics.Moreover, data from the in vivo toxicity study as well as the synthesisof analogs potentially will be of use to the broader scientificcommunity for exploring the role of CDC42 in a range of disease states.

Approach Preliminary Data

Direct Inhibition of CDC42 Diminishes Host Cell Invasion:

Specific pharmacologic inhibition of CDC42 has been examined todetermine whether it is sufficient in the inhibition of host cellinvasion by S. aureus. It was found that pretreatment (0.05 μM, 1 hour)decreased invasion by S. aureus as shown in FIG. 1. Host cellcytotoxicity was undetectable below 30 μM in vitro. The highestconcentration of ML 141 introduced in vivo without detectable toxicitywas 100 mg/kg body weight [BW]. The compound was dissolved inpolyethylene glycol and administered as a single intraperitonealinjection to C57BL/6 mice.

ML 141 Diminishes Intracellular Persistence:

Host Invasion Stimulates Coupling Between CDC42 and PI3Kp85α:

Affinity between the downstream regulator PI3Kp85α and CDC42 is enhancedby GTP-loading of CDC42. Earlier research has demonstrated that S.aureus host cell invasion stimulates GTP-loading of CDC42, raising thepossibility that invasion potentially results in increased couplingbetween these proteins. Within 15 min of host cell invasion, it wasfound that p85α-associated CDC42 had increased (see FIG. 3).

Summary of Preliminary Data

Preliminary research indicates that ML 141 inhibits invasiveness andintracellular persistence potentially through impaired PI3K signaling.The following Experimental Sections examine ML 141 to investigate thetargeting of CDC42 in ameliorating clinically relevant, persistentinfection by S. aureus.

Experimental Section 1: Examine ML 141 in Limiting Persisting S. aureusInfection

Rationale

Increasing evidence supports the hypothesis that S. aureus infectionpersists through pathogenic mechanisms that includes survival withinhost cells. The focus of Section 1 is on exploring new strategies forlimiting infection by this mechanism. It was found that ML 141 decreasedinitial host cell invasion and the number of persistently infecting,intracellular bacteria (see FIGS. 1 and 2). A subset of bacteria thatpersist intracellularly can convert phenotypically to what have beentermed “small colony variants” (SCV). This phenotypically distinctpopulation has been the source of controversy, questioned as alaboratory artifact. However, recent in vivo and clinical evidencesupports the existence of this population and its potential contributionto chronic, recurrent infection. These variants, upon release frompersistently infected host cells, invade new host cells moreaggressively than their parental strain. It is believed that ML 141limits this potential source of recurrent infection by inhibiting theiruptake into new host cells, increasing their clearance by antibiotictherapy and exposure to surveillance by immune cells. Alternatively, ML141 would impair uptake of S. aureus by non-immune cells as a mechanismof clearance.

Study 2 Materials and Methods

Reagents for Cultured Cells:

The following were used at the concentrations and durations indicatedwithin each figure or method described below: paraformaldehyde (ElectronMicroscopy Sciences, Hatfield, Pa.); dimethyl sulfoxide (DMSO) andbovine serum albumin (BSA, Thermo Fisher Scientific, Pittsburgh, Pa.);tryptic soy agar (TSA) and broth (TSB), saponin, lysostaphin,gentamicin, triton, polyethylene glycol (PEG), and formaldehyde(Sigma-Aldrich, St. Louis, Mo.); phosphate buffered saline (PBS),Attachment Factor, M200, Low Serum Growth Supplement (LSGS), rabbitanti-mouse IgG, Alexa Fluor 488 phalloidin, and L-glutamine (LifeTechnologies, Carlsbad, Calif.); XTT (Biotium, Hayward, Calif.); andfetal bovine serum (FBS, Atlanta Biologicals, Lawrenceville, Ga.).4-[3-(4-methoxyphenyl)-5-phenyl-3,4-dihydropyrazol-2-yl]benzenesulfonamide(ML 141) was generously provided by Dr. Jennifer Golden of theUniversity of Kansas Specialized Chemistry Center or was preparedfollowing standard synthetic procedures.

Cell Culture and Compound Treatment:

In one embodiment, human umbilical vein endothelial cells (HUVEC, LifeTechnologies) are cultured in M200 medium supplemented with LSGS. RAW264.7 cells (American Type Culture Collection, ATCC, Manassas, Va.) arecultured in RPMI supplemented with 10% FBS and L-glutamine. All celltypes are maintained at 5% CO₂, 37° C., in 75 cm² vented cap flasks(Thermo-Fisher). For assays, cultured cells are plated at 1×10⁵ cells/mlin 35 mm culture dishes coated with Attachment Factor. The next day,cells are pretreated in culture medium containing the vehicle control orML 141. For compound delivery, ML 141 first is suspended into DMSO orinto PEG at a concentration of 5 mM. The 5 mM solution is diluted to 1mM in the solvent and then diluted to the final concentration for eachexperiment in either serum-containing or serum-free medium. For vehiclecontrol treatment, the same volume of PEG or DMSO as that of ML 141 isadded to medium. The following day, the invasion assay is performed. Forthe shorter duration experiments, compound is added on the same day asthe invasion assay, 1 hour prior to bacteria. Because in vitro dataindicate that ML 141 is a reversible inhibitor, bacteria are addeddirectly to medium containing vehicle control or ML 141.

In another embodiment, HUVEC (Millipore, Billerica, Mass.) are culturedin EndoGRO LS Complete Media (Millipore) and are maintained at 5% CO₂,37° C., in 75 cm² vented cap flasks (Thermo-Fisher). For the invasionassay, cultured cells are plated at 1×10⁴ cells/ml in 96-well culturedishes coated with Attachment Factor. The next day, cells are pretreatedin culture medium containing the vehicle control, ML 141, or ML 141structural analog. For compound delivery, ML 141 and structural analogsfirst are suspended into PEG at a concentration of 5 mM. The 5 mMsolution is diluted to 1 mM in the solvent and then is diluted to thefinal concentration for each experiment in serum containing medium. Forvehicle control treatment, the same volume of PEG as that of ML 141 orof ML 141 structural analog is added to the medium. The following day,the invasion assay is performed. Bacteria are added directly to mediumcontaining vehicle control, ML 141, or the ML 141 structural analog.

Invasion Assay:

Two days prior to the assay, TSB is inoculated with S. aureus (AmericanType Culture Collection #29213) and incubated overnight (225 rpm, 37°C.). Bacteria are subcultured the next day into fresh TSB. On the day ofthe assay, bacteria are pelleted (10000×g, 37° C., 3 min), are washed insaline, are pelleted as above, are resuspended in saline, thenfluorescently labeled by incubation with rabbit anti-mouse IgG AlexaFluor 488 (final concentration 8 μg/ml, RT, 20 min). Protein A, a S.aureus cell surface protein, avidly binds IgG thereby labeling thebacteria. Labeled bacteria are washed twice as above and are resuspendedto 3×10⁸ CFU/ml in saline. Host cells are incubated with the bacteriafor 1 hour (1.2×10⁸ CFU/ml for same-day recovery, 5×10⁶ for recoveryafter 48 hours, 1.4×10⁷ CFU/ml; 5% CO₂, 37° C.). Following infection,extracellular bacteria are removed by extensive washes with PBS andincubation of the host cells with antimicrobials that have limitedmammalian membrane permeability (lysostaphin, 20 μg/ml and gentamicin,50 μg/ml; 45 min for same-day recovery studies, 48 hours for 2-dayrecovery studies; 5% CO₂, 37° C.). To detect the level of infectionusing flow cytometry, cells that have been infected withfluorescently-labeled bacteria are washed extensively with PBS, liftedfrom 96-well plate by incubation with trypsin, washed extensively inFACS buffer (2% BSA/0.1% sodium azide/PBS), fixed (FACS buffercontaining 0.74% formaldehyde), and are counted using an Accuri flowcytometer (BD, Franklin Lakes, N.J.). They are also lifted from culturedishes using cell scrapers, pelleted, fixed (1% BSA/0.74%formaldehyde/PBS), and are counted using an Accuri flow cytometer. Forenumeration of the infecting bacteria, intracellular bacteria arereleased from host cells using 1% saponin/PBS (20 min, 5% CO₂, 37° C.)and serial dilutions are plated on TSA (16 hours, 37° C.). For infectionunder serum free conditions, bacteria first are incubated in FBS (15minutes, RT) followed by extensive washing. The serum incubationprovided extracellular matrix proteins that facilitate invasiveinfection.

Cytotoxicity Assay:

HUVEC are plated at a density of 1.2×10⁴ cells/ml into 96-well dishescoated with Attachment Factor. The next day, cells are pretreated withthe vehicle control DMSO or with ML 141 (18 hours). An XTT-reducingassay is performed and absorbance read at 490 nanometers using a Bio-Radplate reader.

Assessment of Bactericidal Activity:

HUVEC are pretreated with the vehicle control DMSO or with ML 141 (18hours) then incubated with 5×10⁶ CFU/ml (1 hour). Following infection,the medium from each plate is removed, is serially diluted into saline,and is plated on fresh blood agar plates. The next day, colony countsare performed and hemolysis is recorded.

Immunofluorescence:

HUVEC are plated at 1×10⁵ cells/ml into 35 mm glass-bottom dishes(MatTek, Ashland, Mass.) are coated with Attachment Factor, arepretreated, and are infected (1.2×10⁸ CFU) as described above. Followinginfection, cells are washed with 1×PBS, fixed (4% paraformaldehyde/PBS,30 min), permeabilized, blocked (0.1% Triton, 1% bovine serum albumin,30 min), and incubated with Alexa Fluor 488 phalloidin (1:40). Confocalimages are acquired using an inverted Zeiss Axiovert200 microscopeequipped with a plan-apochromat 40×, 1.2 NA water immersion lens withcorrection collar and LSM 5 Pascal scan head. Alexa 488 is excited bythe 488 nanommeter Ar laser line and is detected using a 505-530nanometer bandpass filter. Z-sectioning and frame size are set toNyquist sampling. Maximum pixel projections from the Z-stacks aregenerated and are analyzed for actin morphology.

Statistical Analyses:

Normally distributed data are analyzed by Student's t-test when thecomparison was limited to 2 groups or by one-way ANOVA followed byStudent-Newman-Keuls post-hoc analysis when 3 or more groups werecompared (Sigma Stat, Systat, Point Richmond, Calif.). Differencesbetween groups were considered statistically significant at p<0.05.

Results and Discussion

ML 141 Limits Host Cell Invasion.

Earlier work had indicated that CDC42 activity is stimulated by S.aureus host cell invasion and that limiting CDC42 function using agenetic strategy led to a reduction in S. aureus invasiveness. Todetermine whether specific, pharmacologic inhibition of CDC42 issufficient to inhibit invasion by S. aureus, HUVEC are pretreated (18hours) with ML 141 (10.0 μM) or with the vehicle control PEG and areinfected for 1 hour. ML 141 treatment decreased invasion by more than80% (p≦0.001 by Student's t-test, FIG. 4, Panel A). When ML 141 isdelivered in DMSO rather than in PEG, inhibition nears 50% (Panel B).Inhibition is detectable when treatment was reduced from 18 hours to 1hour under serum-starved conditions (FIG. 5). ML 141 inhibited invasionin all cell types examined (HUVEC, HEK, U-87 MG, RAW and A549).

The number of uninfected cells is greatest when ML 141 has beendelivered in PEG rather than in DMSO (FIGS. 4A-1-4B-2), suggesting thatPEG enhanced the delivery of ML 141. PEG is a well-characterized,hydrophilic polymer that can increase the delivery of hydrophobiccompounds into mammalian cells. The increased effectiveness of ML 141when delivered in PEG may be attributable to an increase in thesolubility of ML 141. Evidence supporting this concept is thatsuspension in PEG enhances the solubility of celecoxib, a COX-2inhibitor that is structurally similar to ML 141. That PEG generally iswell-tolerated with minimal toxicity is supported by the finding thatcytotoxicity was not observed at the concentrations used in the invasionassay (FIGS. 6A-6B).

Cytotoxicity and Bactericidal Activity were not Detected.

To determine whether host or bacterial cell death contributed to thedecreases in recovered bacteria, cytotoxicity and bactericidal activityare assessed. Cytotoxicity is not detectable in HUVEC treated overnightin ML 141 in the PEG solvent (FIG. 6A; p≧0.05 by one-way ANOVA).Bactericidal activity was not detected (FIG. 6B). These findings suggestthat the decreases in infected host cells and in recovered bacteria areattributable to diminished host cell invasion rather than to host celldeath or to bactericidal activity of the compound.

Intracellular Populations Remain Suppressed Over Time.

Intracellular bacterial populations can continue to proliferate,therefore, the intracellular bacterial population within ML 141-treatedhost cells was examined to determine whether it returned to controllevels over time. HUVEC are pretreated with vehicle control or with ML141 (1.0 μM, 18 hours), infected for 1 hour, and bacterial levels withinhost cells are assessed at 48 hours post infection. These experimentsare pursued in endothelial cells as a model system for growth ofintracellular reservoirs and because of the central role played by theendothelial cell in the pathogenesis of endocarditis. Treatment with ML141 diminished the number of viable bacteria recovered 48 hourspost-infection (FIG. 7).

Intracellular persistence by S. aureus enables the pathogen to evadeantibiotic therapies and surveillance by immune cells. Thisintracellular residency establishes bacterial reservoirs as sources ofchronic infection. Bacteria that persist intracellularly can convertphenotypically so that upon release from aged cells, the population isable to invade new host cells more aggressively. This passage from oldercells into new cells is believed to contribute to chronic, recurrentinfection. Taken together, findings that ML 141 limits populations ofpersisting bacteria with limited cytotoxic or bactericidal activitypoints to the possible usefulness of targeting CDC42 to augment currenttherapeutic approaches for chronic, recurrent infection.

RSM series as inhibitory compounds for Staphylococcus aureus inendothelial cells

The scope of this disclosure is to: 1) synthesize a series of ML 141structural analogs; 2) assess these structural analogs using an invasionassay; and 3) characterize further the pharmacology of ML 141.

Experimental Section 3: Develop analogs to increase ML 141 efficacy andpotency

Rationale

ML 141 demonstrates specificity for CDC42, yet compound usefulness maybe limited by its solubility. The goal of this Section is to design andsynthesize novel analogs with improved solubility that efficientlyinhibit GTP-loading of CDC42 and S. aureus invasion. The modifiedanalogs are based on the core structure ML 141 as illustrated in Scheme0. Modifications will be made at specific locations on the corearomatics at the three and/or five positions of the pyrazoline core inan attempt to increase hydrophilicity. The aromatic pyridineheterocycles or ether and alcohol groups will increase aqueoussolubility and these groups are shown as generic spheres.

Study 1

Addition of Small Ether or Alcohol Groups or Larger Polyethylene GlycolAppendages to Specific Locations on the Core Aromatics in an Attempt toIncrease Hydrophilicity.

The proposed synthesis commences with the reaction of4-hydroxybenzaldehyde with alkylating agents such as 2-chloroethanol,2-(2-chloroethoxy)ethanol, 2-[2-(2-chloroethoxy)ethoxy]ethanol (R═H) ortheir corresponding methyl ethers (R═CH₃) in sodium hydroxide. Theresulting ether synthesis provides a more hydrophilic benzaldehyde asshown in Scheme 1. The attachment of the small ether or alcohol groupsor larger polyethylene glycol appendages to the starting acetophenonehas also been proven. The mixed aldol condensation of acetophenone and4-methoxybenzaldehyde for the synthesis of ML 141 can be adapted for theconstruction of proposed analogs. Following literature precedent, thereaction of substituted acetophenones with substituted benzaldehydeswill prepare the required chalcones as shown in Scheme 1. Thesechalcones will then be condensed and cyclized with4-hydrazinobenzenesulfonamide, which is easily prepared as thehydrochloride salt from sulfanilamide. The appendages can be extended toa longer polyethylene glycol (PEG) substituent if desired. It is wellknown that the addition of PEG chains to organic compounds can increaseaqueous solubility without fear to biological safety. The appendages canbe extended to a PEG substituent. Indeed, several pharmaceutical drugshave made it to market or are currently in clinical trials withhydrophilic appendages.

It is expected that replacement of the 4-methoxy substituent on thephenyl substituent at the five position of the pyrazoline withfunctionalized alkoxy substituents or addition of the functionalizedalkoxy substituents to the phenyl moiety at the three position providesnovel structural analogs of ML 141 with equivalent biological activitybut improved solubility.

Results and Discussion

ML 141 Structural Analogs

The chemistry for the synthesis of ML 141 structural analogs is shown inScheme 2. Hydrazine hydrochlorides are commercially available orprepared from their corresponding amines through diazotization followedby stannous chloride reduction. The prerequisite chalcones are preparedvia aldol condensation from commercially available ketones with aromaticaldehydes. The general procedure is described as follows: the aldehyde(7.0 mmol) and ketone (7.0 mmol) are dissolved in ethanol (10-25 mL),the solution cooled to 0° C., and then 40% NaOH gradually added dropwiseuntil precipitation commenced. The cold mixture is stirred untilprecipitation is complete and the product is then collected by vacuumfiltration. The pyrazolines are synthesized as follows: the chalcone(1.0 mmol) and hydrazine hydrochloride (1.0 mmol) are dissolved inethanol (25 mL) and a catalytic amount of sodium acetate is added (0.05mmol). The reaction is then heated at reflux until determined completeby TLC (8-30 h). The solution is concentrated to half volume and then iscooled to produce solid product, which is then collected by vacuumfiltration. If no solid is obtained, the remaining solvent is removedvia rotoevaporation and the solid is taken up in ethyl acetate, washedwith water, dried with sodium sulfate, filtered, and solvent evaporatedto obtain crude product. Purification of crude product is achieved viarecrystallization or column chromatography on silica gel.

Structural analogs of ML 141 (RSM 04, 05, 06, 07, 11-18), shown in FIGS.14A through 14L, are prepared specifically fromp-sulfamylphenylhydrazine hydrochloride and the chalcones of methylketones and aromatic aldehydes as shown in Scheme 2. Structural analogRSM 19 is prepared from p-sulfamylphenylhydrazine hydrochloride and thechalcone of propiophenone and 4-methoxybenzaldehyde. The structuralanalogs are purified by standard means and characterized by IR and NMRspectroscopy.

ML 141 Structural Analogs Inhibit Host Cell Invasion

To examine whether structural analogs of ML 141 inhibit host cellinvasion by S. aureus, HUVEC are incubated with structural analogcompound (10 μM) or with an equimolar amount of ML 141 and are assayedagainst vehicle control treated infected samples. The examinedstructural analogs all inhibit host cell invasion as compared to thecontrol (Tables 1 and 2), as shown by significantly lower meanfluorescence when compared to the PEG control.

Referring to Table 1, inhibition by RSM 05, RSM 06, RSM 07, RSM 11, RSM12, RSM 13, RSM 17, RSM 19, RSM 20, RSM 21, and RSM 26 is similar toinhibition by ML 141. RSM 04, RSM 06, RSM 15, and RSM 16 inhibitedinvasion more than ML 141. RSM 05, RSM 15, and RSM 16 have GTP IC₅₀concentrations in the same 2-4 μM range as ML 141, and RSM 07 and RSM 21are only slightly higher.

Referring to Table 2, inhibition by RSM 27, RSM 28, RSM 29, RSM 30, RSM31, RSM 32 and RSM 33 is similar to inhibition by ML 141.

TABLE 1 ML 141 structural analogs inhibit host cell invasion. Humanumbilical vein endothelial cells (HUVEC) are incubated with ML 141 (10μM), with ML 141 structural analogs (designated RSM 01-26; 10 μM), orwith vehicle control polyethylene glycol (PEG, 1%), 18-20 hours prior toinfection with fluorescently labeled Staphylococcus aureus (1 hour, 5%CO₂, 37° C.). Extracellular bacteria are removed using lysostaphin andgentamicin. Internalized bacteria are detected using flow cytometry(*less than vehicle control, ^(†)greater than ML 141, ^(#)less than ML141, p ≦ 0.05; ^(‡)not different than ML 141, p > 0.05; n =5/treatment). With regard to the GTP binding column, BODIPY-FL-GTPbinding to CDC42 was assessed for a subset of the RSM series. Forcomparison, the reported IC₅₀ of ML 141 is 2-4 μM. Inhibition ofBODIPY-FL-GTP binding to Rab7 was not detected when tested up to 100 μM.Internalized bacteria (% control ± SEM) Structural analog ML 141 GTPbinding (IC₅₀) RSM 04 11 ± 1%*^(#) 35 ± 5%* RSM 05 38 ± 3%*^(‡) 42 ± 3%*3.4 μM RSM 06 23 ± 1%*^(#) 39 ± 5%* 10.1 μM  RSM 07 34 ± 2%*^(‡) 36 ±3%* 6.2 μM RSM 11 61 ± 2%*^(‡) 57 ± 6%* RSM 12 57 ± 3%*^(‡) 47 ± 4%* RSM13 62 ± 5%*^(‡) 63 ± 6%* RSM 14 71 ± 3%*^(†) 51 ± 3%* RSM 15 45 ±1%*^(#) 53 ± 3%* 2.2 μM RSM 16 63 ± 4%*^(#) 89 ± 3%* 3.7 μM RSM 17 67 ±8%*^(‡) 66 ± 1%* RSM 18 76 ± 7%*^(†) 52 ± 3%* RSM 19 61 ± 2%*^(‡) 58 ±3%* 13.5 μM  RSM 20 61 ± 4%*^(‡)  50 ± 14%* 46.5 μM  RSM 21 79 ± 3%*^(‡)61 ± 3%* 5.5 μM RSM 26 40 ± 3%*^(‡) 58 ± 3%*

TABLE 2 ML141 and RSM structural analogs decrease intracellularinfection. Human umbilical vein endothelial cells (HUVEC) were incubated(18-20 h) with ML141 (10 μmol/L), RSM structural analog (10 μmol/L), ordimethyl sulfoxide (DMSO), infected with Staphylococcus aureus, andextracellular bacteria removed using gentamicin and lysostaphin.Bacteria per host cell were counted in 100 cells from randomly selectedfields using florescence microscopy. Intracellular bacteria/cell (±SEM)Structural analog ML 141 DMSO RSM 27 4.4 ± 0.5* 3.4 ± 0.4* 8.0 ± 0.8 RSM28 1.4 ± 0.3* 1.6 ± 0.2* 6.2 ± 0.8 RSM 29 2.4 ± 0.4* 1.6 ± 0.2* 6.2 ±0.8 RSM 30 1.7 ± 0.3* 1.2 ± 0.3* 7.3 ± 0.8 RSM 31 1.4 ± 0.4* 1.2 ± 0.3*7.3 ± 0.8 RSM 32 5.5 ± 0.4* 5.6 ± 0.4* 10.7 ± 0.8  RSM 33 5.4 ± 0.4* 5.6± 0.4* 10.7 ± 0.8  *Less than DMSO control by one-way ANOVA followed byNewman-Keuls multiple comparison test.

TABLE 3 Molecular structure of ML 141 and related RSM pyrazolineanalogs.

ID Ar R Ar′ R′ ML 141 Ph H 4-MeOPh H RSM 04 4-MeOPh H 4-ClPh H RSM 05 PhH 3,4-(OCH₂O)Ph H RSM 06 4-MeOPh H 3,4-(OCH₂O)Ph H RSM 07 4-MeOPh H Ph HRSM 11 Ph H 4-MeOCH₂CH₂OPh H RSM 12 4-MeOPh H 4-MeOCH₂CH₂OPh H RSM 13 PhH 3,4,5-MeOPh H RSM 14 4-MeOPh H 3,4,5-MeOPh H RSM 15 Ph H 3,4-MeOPh HRSM 16 4-MeOPh H 3,4-MeOPh H RSM 17 4-MeOCH₂CH₂OPh H 4-MeOPh H RSM 184-MeOCH₂CH₂OPh H 4-MeOCH₂CH₂OPh H RSM 19 Ph Me 4-MeOPh H RSM 204-MeOCH₂CH₂OPh H 4-ClPh H RSM 21 Ph-(2-CH₂)— 4-MeOPh H RSM 26 Ph H4-MeOPh Me RSM 27^(#) Ph — 4-MeOPh — RSM 28 Ph H 4-Me₂NPh H RSM 29 Ph H4-MeCONHPh H RSM 30 3-MeCONHPh H 4-MeOPh H RSM 31 4-MeCONHPh H 4-MeOPh HRSM 32 4-EtCONHPh H 4-MeOPh H RSM 33 3-EtCONHPh H 4-MeOPh H ^(#)RSM 27is acyclic hydrazine in place of pyrazoline:

TABLE 4 Assessment of cytotoxicity. Human umbilical vein endothelialcells were pretreated (18-20 h) with vehicle control, with ML 141 (10μM), or with structural analog at equimolar concentration and viabilityassessed by propidium iodide (P1) exclusion (**greater than vehiclecontrol, p < 0.01 by Student's t-test; n = 3/treatment). Meanfluorescence intensity (×10⁴ ± SEM) Vehicle control Compound (10 μM) ML141 1.7 ± 0.1 2.1 ± 0.3 RSM 04 1.9 ± 0.2  4.6 ± 0.2** RSM 05 2.6 ± 0.83.9 ± 0.2 RSM 06 2.4 ± 0.3 2.8 ± 0.1 RSM 07 1.5 ± 0.8 2.5 ± 0.4 RSM 153.6 ± 0.8 4.0 ± 0.4 RSM 16 1.9 ± 0.2 1.4 ± 0.2 RSM 19 2.0 ± 0.3  2.6 ±0.01 RSM 20 2.6 ± 0.8 2.1 ± 0.2 RSM 21 2.4 ± 0.3  2.8 ± 0.09 RSM 26 13.3± 1.5   26.8 ± 2.1**

TABLE 5 Assessment of cytotoxicity. RAW 264.7 cells were plated andtreated as described for the invasion assay (Table 2). Followingpretreatment (18-20 h), RAW cells were lifted from plates using cellscrapers, washed extensively in FACS buffer, incubated briefly withpropidium iodide (Sigma-Aldrich, 0.5 mg/mL), and fluorescence detectedimmediately using an Accuri C6 flow cytometer. Mean fluorescenceintensity (×10³ ± SEM) Vehicle control Compound (10 μM) ML 141 2.2 ± 0.22.5 ± 0.3 RSM 27 1.1 ± 0.0 1.0 ± 0.1 RSM 28 9.8 ± 1.2  11 ± 2.0 RSM 293.1 ± 0.7 4.2 ± 0.3 RSM 30 2.9 ± 0.5 2.4 ± 0.4 RSM 31 1.6 ± 0.1 2.4 ±0.3 RSM 32 1.3 ± 0.1 2.8 ± 1.1 RSM 33 1.3 ± 0.1 2.9 ± 1.1

TABLE 6 Assessment of bactericidal activity. 1.2 × 10⁸ colony formingunits (CFU) of Staphylococcus aureus were incubated with compound orwith vehicle control in human umbilical vein endothelial cell media (1h, 5% CO₂, 37° C.). Serial dilutions were plated onto tryptic soy agar,incubated (18-20 h, 37° C.), colonies enumerated, and CFU/ml determined(*less than control, p < 0.05 by Student's t-test, n = 3/treatment). CFU(×10⁸ ± SEM) Vehicle control Compound (10 μM) ML 141 9.8 ± 1.5 8.8 ± 2.9RSM 04 6.0 ± 1.3 3.8 ± 0.8 RSM 05 12.1 ± 0.5  8.0 ± 1.7 RSM 06 3.4 ± 0.54.9 ± 1.6 RSM 07 13.8 ± 1.6  11.5 ± 1.1  RSM 15 9.0 ± 0.6 10.1 ± 1.0 RSM 16 8.6 ± 0.2 8.9 ± 1.9 RSM 19 10.7 ± 0.6  9.4 ± 1.0 RSM 20 12.5 ±0.6  10.0 ± 0.2  RSM 21 15.0 ± 1.1  15.0 ± 1.1  RSM 26 9.8 ± 1.1  0.8 ±1.1*

TABLE 7 Assessment of bactericidal activity. 3.2 × 10⁶ colony formingunits (CFU) of Staphylococcus aureus were incubated with compound (ML141 or analog) or with vehicle control in Dulbecco's Modified Eagle'sMedium supplemented with 10% fetal bovine serum (1 h, 5% CO₂, 37° C.).Serial dilutions were plated onto tryptic soy agar, incubated (18-20 h,37° C.) colonies enumerated, and CFU/ml determined (p < 0.05 byStudent's t-test, n = 3-5/treatment). CFU/mL (×10⁹ ± SEM) 20 mM Vehiclecontrol Compound ML141 0.9 ± 0.07 0.9 ± 0.09 RSM 27 2.6 ± 0.2 2.4 ± 0.6RSM 28 1.5 ± 0.07 1.8 ± 0.05 RSM 29 1.3 ± 0.3 1.4 ± 0.1 RSM 30 1.4 ± 0.11.5 ± 0.02 RSM 31 1.3 ± 0.2 0.9 ± 0.09 RSM 32 2.7 ± 0.2 2.4 ± 0.3 RSM 331.4 ± 0.2 1.3 ± 0.04

Depolymerization of Actin Stress Fibers During Infection is Limited byML 141

When actin stress fibers depolymerize, the actin monomers reorganize atthe cell membrane to facilitate endocytic uptake. The process appears tobe partially regulated by the small GTPase CDC42, indicated by thefinding that in cells lacking active CDC42, depolymerization is limitedas is endocytic uptake. Research had found previously that host cellinvasion by S. aureus stimulates the depolymerization of actin stressfibers. Inhibition of this depolymerization by LY294002, an inhibitor ofphosphoinositide 3-kinase activity, was associated with decreasedinvasion, suggesting that the depolymerization of stress fibers andredistribution of actin at the cell membrane facilitates invasion. Nextwas examined whether the specific inhibition of CDC42 using ML 141 wouldlimit depolymerization during infection. In response to infection,stress fibers depolymerized in 75% of vehicle treated cells yet remainedintact in ML 141 treated cells (FIG. 8). This finding indicates that ML141 blunts the depolymerization of actin stress fibers during infection.The finding suggests that an underlying mechanism for ML 141 inhibitionof invasion is due in part to the limited redistribution of actinrequired for endocytic uptake of pathogenic S. aureus. Two of the ML 141derivatives were selected to evaluate whether either compound limitedthe redistribution of actin. Both RSM 06 and RSM 16 limited actindepolymerization during invasion. These findings suggest that theinhibition of invasion by RSM 6 and by RSM 16 is due in part to thelimited redistribution of actin.

Implications

ML 141 and its structural analogs provide a unique tool for exploringthe role of CDC42 in mediating host cell invasion. Taken together, theinvestigation into ML 141 and its structural analogs has the potentialto provide evidence that supports future compound development for thetreatment of invasive infection. Alternatively, findings may open newdirections for research into the role of this small-GTPase in hostimmune responses. Either outcome will impact the evaluation of treatmentstrategies that address infection at the level of the host, elucidatecellular processes under the regulation of CDC42, and expand upon thecharacterization of ML 141 and its structural analogs.

NMR Data

RSM 04

¹H NMR (300 MHz, CDCl₃) δ 3.14 (dd, J=5.8 Hz, 17.0 Hz, 1H), 3.85 (s,3H), 3.88 (dd, J=12.1 Hz, J=17.0 Hz, 1H), 4.59 (s, 2H), 5.31 (dd, J=5.8Hz, 12.1 Hz), 6.93 (d, J=12.4 Hz, 2H), 7.03 (d, J=9.1 Hz, 2H), 7.19 (d,J=8.6 Hz, 2H), 7.31 (d, J=8.6 Hz, 2H), 7.67 (d, J=4.7 Hz, 2H), 7.69 (d,J=5.0 Hz, 2H)

RSM 05

¹H NMR (300 MHz, CDCl₃) δ 3.11 (dd, J=5.8 Hz, 17.6 Hz, 1H), 3.80 (dd,J=12.1 Hz, 17.6 Hz, 1H), 5.22 (dd, J=5.8 Hz, 12.1 Hz, 1H), 5.53 (s, 2H),5.86 (s, 2H), 6.61 (s, 1H), 6.69 (s, 2H), 7.02 (d, J=8.3 Hz, 2H),7.40-7.34 (m, 3H), 7.69-7.65 (m, 4H)

RSM 06

¹H NMR (300 MHz, CDCl₃) δ 3.16 (dd, J=5.8 Hz, 17.3 Hz, 1H), 3.83 (dd,J=12.4 Hz, 17.3 Hz, 1H), 3.85 (s, 3H), 5.24 (dd, J=5.8 Hz, 12.4 Hz, 1H),5.93 (s, 2H), 6.67 (s, 1H), 6.76 (s, 2H), 6.93 (d, J=9.1 Hz, 2H), 7.07(d, J=9.1 Hz, 2H), 7.67 (d, J=4.5 Hz, 2H), 7.69 (d, J=4.5 Hz, 2H)

RSM 07

¹H NMR (300 MHz, DMSO-D₆) δ 3.16 (dd, J=5.0 Hz, 17.6 Hz, 1H), 3.80 (s,3H), 3.95 (dd, J=12.1 Hz, 17.6 Hz, 1H), 5.60 (dd, J=5.0 Hz, 12.1 Hz,1H), 7.06-6.99 (m, 6H), 7.39-7.26 (m, 5H), 7.57 (d, J=9.1 Hz, 2H), 7.74(d, J=8.8 Hz, 2H)

RSM 11

¹H NMR (400 MHz, CDCl₃) δ 3.18 (dd, J=17.2 Hz, 5.9 Hz, 1H), 3.42 (s,3H), 3.71 (t, J=4.8 Hz, 2H), 3.86 (dd, J=17.6 Hz, 12.6 Hz 1H), 4.06 (t,J=4.8 Hz, 2H) 4.69 (s, 2H), 5.30 (dd, J=12.1 Hz, 5.8 Hz, 1H), 6.86 (d,J=8.4 Hz, 2H), 7.07 (d, J=8.8 Hz, 2H), 7.15 (d, J=8.8 Hz, 2H), 7.35-7.43(m, 3H), 7.67 (d, J=8.8 Hz, 2H), 7.73 (dd, J=7.6 Hz, 1.4 Hz, 2H)

RSM 12

¹H NMR (400 MHz, CDCl₃) δ 3.16 (dd, J=17.3 Hz, 5.8 Hz, 1H), 3.43 (s,3H), 3.72 (t, J=5.0 Hz, 2H) 3.79-3.89 (m, 4H), 4.08 (t, J=4.7 Hz, 2H),4.59 (s, 2H), 5.28 (dd, J=12.1 Hz, 5.8 Hz, 1H), 6.91 (dd, J=15.7 Hz, 8.8Hz, 4H), 7.05 (d, J=8.8 Hz, 2H), 7.16 (d, J=8.5 Hz, 2H), 7.68 (dd, J=9.1Hz, 4.1 Hz, 4H)

RSM 13

¹H NMR (300 MHz, CDCl₃) δ 3.23 (dd, 6.6 Hz, 17.6 Hz, 1H), 3.80 (s, 6H),3.83 (s, 3H), 3.89 (dd, J=12.4 Hz, 17.6 Hz, 1H), 5.25 (dd, J=6.6 Hz,12.4 Hz, 1H), 6.46 (s, 2H), 7.12 (d, J=8.8 Hz, 2H), 7.47-7.41 (m, 3H),7.78-7.74 (m, 4H)

RSM 14

¹H NMR (300 MHz, CDCl₃) δ 3.20 (dd, J=6.6 Hz, 17.6 Hz, 1H), 3.80 (s,6H), 3.83 (s, 3H), 3.85 (s, 3H), 3.85 (dd, J=12.1 Hz, 17.6 Hz, 1H), 5.21(dd, J=6.6 Hz, 12.1 Hz, 1H), 6.46 (s, 2H), 6.94 (d, J=9.6 Hz, 2H), 7.09(d, 8.8 Hz, 2H)

RSM 15

¹H NMR (300 MHz, DMSO-D₆) δ 3.19 (dd, J=5.5 Hz, 17.6 Hz, 1H), 3.69 (s,3H) 3.72 (s, 3H), 3.94 (dd, J=12.1 Hz, 17.6 Hz, 1H), 5.54 (dd, J=5.5 Hz,12.1 Hz, 1H), 6.69-6.67 (m, 1H), 6.88 (d, J=8.1 Hz, 1H), 6.94 (d, J=2.1Hz, 1H), 7.09 (d, J=8.6 Hz, 2H), 7.48-7.39 (m, 3H), 7.59 (d, J=9.2 Hz,2H), 7.79 (d, J=6.6 Hz, 2H)

RSM 16

¹H NMR (300 MHz, DMSO-D₆) δ 3.15 (dd, J=5.5 Hz, 17.6 Hz, 1H), 3.68 (s,3H) 3.71 (s, 3H), 3.79 (s, 3H), 3.89 (dd, J=12.1 Hz, 17.6 Hz, 1H), 5.84(dd, J=5.5 Hz, 12.1 Hz, 1H), 6.68-6.67 (m, 1H), 6.87 (d, J=8.1 Hz, 1H),6.93 (d, J=2.1 Hz, 1H), 7.07-6.99 (m, 6H), 7.57 (d, J=9.2 Hz, 2H), 7.72(d, J=7.8 Hz, 2H)

RSM 17

¹H NMR (400 MHz, CDCl₃) δ 3.15 (dd, J=17.6 Hz, 5.8 Hz, 1H), 3.46 (s,3H), 3.75-3.78 (m. 5H), 3.83 (dd, J=17.2 Hz, 12.1 Hz, 1H), 4.15 (t,J=4.8 Hz, 2H), 4.60 (s, 2H), 5.28 (dd, J=12.1 Hz, 5.5 Hz, 1H), 6.85 (d,J=8.4 Hz, 2H), 6.95 (d, J=8.8 Hz, 2H), 7.05 (d, J=8.8 Hz, 2H), 7.16 (d,J=8.4 Hz, 2H), 7.67 (d, J=8.8 Hz, 4H)

RSM 18

¹H NMR (400 MHz, CDCl₃) δ 3.15 (dd, J=17.2 Hz, 5.9 Hz, 1H), 3.43 (s,3H), 3.46 (s, 3H), 3.72 (t, J=4.8 Hz, 2H), 3.77 (t, J=4.8 Hz, 2H) 3.83(dd, J=17.2 Hz, 12.1 Hz, 1H), 4.08 (t, J=4.8 Hz, 2H), 4.15 (t, J=4.4 Hz,2H), 4.62 (s, 2H), 5.27 (dd, J=12.1 Hz, 5.9 Hz, 1H), 6.88 (d, J=8.8 Hz,2H), 6.96 (d, J=8.8 Hz, 2H), 7.05 (d, J=8.8 Hz, 2H), 7.15 (d, J=8.8 Hz,2H), 7.67 (dd, J=9.2 Hz, 2.6 Hz, 4H)

RSM 19

¹H NMR (400 MHz, CDCl₃) δ 0.84 (d, J=7.7 Hz, 3H), 3.81 (s, 3H),3.99-4.08 (m, 1H), 4.64 (s, 2H) 5.30 (d, J=11.4 Hz, 1H) 6.87-6.90 (m,2H), 7.08 (d, J=8.8 Hz, 2H), 7.14-7.15 (m, 2H), 7.36-7.45 (m, 3H), 7.67(d, J=8.8 Hz, 2H), 7.77 (dd, J=8.4 Hz, 1.5 Hz, 2H)

RSM 20

¹H NMR (400 MHz, CDCl₃) δ 3.15 (dd, J=17.2 Hz, 5.9 Hz, 1H), 3.47 (s,3H), 3.77 (t, J=4.8 Hz, 2H), 3.87 (dd, J=17.2 Hz, 12.1 Hz, 1H) 4.16 (t,J=4.4 Hz, 2H), 4.60 (s, 2H), 5.31 (dd, J=12.1 Hz, 5.9 Hz, 1H), 6.96 (d,J=8.8 Hz, 2H), 7.03 (d, J=9.2 Hz, 2H), 7.19 (d, J=8.4 Hz, 2H), 7.31 (d,J=8.4 Hz, 2H), 7.68 (t, J=9.2, 4H). 13C NMR (100 MHz, DMSO-de) δ 43.5,56.6, 58.8, 62.1, 67.7, 70.9, 112.4, 115.3, 124.9, 127.8, 128.3, 129.6,132.7, 133.4, 141.3, 146.5, 150.2, 160.1

RSM 21

¹H NMR (400 MHz, CDCl₃) δ 2.23 (dd, J=16.1 Hz, 7.7 Hz, 1H), 2.91 (dd,J=15.7 Hz, 9.2 Hz, 1H), 3.72 (s, 3H), 4.18-4.24 (m, 1H), 4.61 (s, 2H),5.55 (d, J=11.0 Hz, 1H), 6.75-6.77 (m, 2H), 6.94-7.03 (m, 4H), 7.21 (d,J=6.1 Hz, 1H), 7.28-7.34 (m, 2H), 7.67 (d, J=9.2 Hz, 2H), 7.76 (d, J=8.4Hz, 1H). 13C NMR (100 MHz, DMSO-de) δ 54.8, 55.5, 56.7, 66.9, 112.6,114.8, 122.7, 126.8, 127.2, 127.7, 128.3, 131.0, 133.1, 147.5, 152.2,159.2, 164.2

RSM 26

¹H NMR (300 MHz, CDCl₃) δ 1.87 (s, 3H), 3.47 (s, 2H), 3.82 (s, 3H), 4.60(s, 2H), 6.90 (d, J=8.8 Hz, 2H), 7.06 (d, J=9.1 Hz, 2H), 7.45-7.31 (m,5H), 7.62 (d, J=9.1 Hz, 2H), 7.73 (d, J=9.6 Hz, 2H)

Experimental Section 4: Inhibition of CDC42 Limits S. aureus Infection

Rationale

Selective small molecule inhibition of CDC42 may disrupt cellularprocesses used by S. aureus to gain host cell entry and is a druggabletarget in the treatment of invasive infection.

Materials and Methods

Reagents.

Reagents were used at the concentrations and durations indicated withineach figure legend or method described below: human fibronectin(Sigma-Aldrich, St. Louis, Mo.); all remaining reagents were sourced asdescribed in Experimental Section 1 above.

Structural Analog Synthesis.

General procedure for pyrazolines known as RSM structural analogs(Bashir et al., 2011). Equimolar equivalents of the correspondingchalcone (see below) and p-sulfamylphenylhydrazine hydrochloride weredissolved in 95% ethanol (25 mL/mmol) and a catalytic amount of sodiumacetate was added. The mixture was then refluxed for 24 hr. The reactionmixture was concentrated to about ⅓ of the volume via simpledistillation. The reaction was then cooled to room temperature and theproduct was isolated via vacuum filtration. When necessary, the productwas recrystallized with ethanol or purified by flash chromatography onsilica gel.

General Procedure for Chalcone Synthesis.

The corresponding benzaldehyde (1.0 equiv.) and the correspondingacetophenone (1.0 equiv.) derivatives were dissolved in 95% ethanol (1.5mL/mmol) and cooled to 0° C. in an ice bath. A solution of 40% sodiumhydroxide (up to 0.5 mL/mmol) was then slowly added drop wise over 1 hor until solid begins to precipitate. The mixture was left to stir at 0°C. for another 30 min and the product was isolated via vacuumfiltration. The crude solid was washed with cold 95% ethanol and wasrecrystallized using 95% ethanol. Chalcone used for the preparation ofRSM 26 was prepared via cuprate addition to the conjugated ynone.

General Procedure for Alkylation of Phenols.

4-Hydroxyacetopheone or 4-hydroxybenzaldehyde (1.00 equiv.) was added toa solution of 2-chloroethyl methyl ether or 2-(2-tosylethoxy)ethylmethyl ether (1.20 equiv.) and potassium carbonate (1.20 equiv.) in DMF(1.5 mL/mmol). The reaction mixture was heated to 60° C. and left tostir for 16 hours. To the cooled mixture was added water (30 mL) andextracted with ethyl acetate (3×30 mL). The combined organic layers werethen washed with 1M NaOH (1×30 mL) water (3×30 mL) and brine (1×30 mL)and dried over magnesium sulfate. The solvent was removed in vacuo.

Transmission Electron Microscopy.

HUVEC were plated onto glass coverslips at 3×10/ml, pretreated andinfected at a multiplicity of infection (MOI) of 30 for 2 h.Extracellular bacteria were removed and cells incubated for anadditional 24 h in media containing antibiotics. Cells were immersionfixed with 2.5% glutaraldehyde/2.0% paraformaldehyde/2 mM calcium/1 mMmagnesium (15 min, twice), washed extensively with PBS, post-fixed with1.0% osmium tetroxide/1.5% potassium ferrocyanide in 0.1 M sodiumcacodylate buffer, dehydrated in a graded ethanol series followed bypropylene oxide, and embedded in EMBed812 (Electron MicroscopySciences). Serial ultrathin sections were cut, collected onto Pioloformslot grids, and counterstained with aqueous uranyl acetate and Reynold'slead citrate (30 min each). Electron micrographs were obtained at 120 KVusing a JEOL JEM-1400 equipped with a Gatan Ultrascan 1000XP camera.

Flow Cytometry.

To examine β1 surface expression, recycling of the integrin wasterminated (4° C., 15 min) and HUVEC lifted from plates using cellscrapers. After washing in FACS buffer (2% BSA/0.1% sodium azide/PBS),cells were incubated with PE-conjugated anti-β1 (BD; 30 min, on ice),washed, fixed in FACS buffer containing 4% paraformaldehyde, andanalyzed using an Accuri C6 flow cytometer (BD).

Fibronectin Adhesion Assay.

HUVEC were plated and pretreated as described for the invasion assay.96-well, non-tissue culture treated plates (Sarstedt, Newton, N.C.) werecoated with fibronectin (20 μg/ml; 2 h; 37° C.), washed with PBS, andblocked with bovine serum albumin (2%, 30 min, RT). HUVEC were liftedfrom the 35 mm dishes using cell scrapers, 100 μl counted fornormalization using the Accuri C6, and 200 μl transferred to the 96-wellplate (2 h, 5% CO₂, 37° C.). After extensive washes in PBS, cells wereremoved from the plate using trypsin, washed in FACS buffer, fixed, and100 μl counted. These cell counts were normalized to those that had beentaken prior to plating on the fibronectin-coated plate.

Methods not explicitly described in Experimental Section 4 wereperformed as described in Experimental Section 1.

Results and Discussion

Small Molecule Inhibition of CDC42 Limits Host Cell Invasion by S.aureus.

HUVEC were incubated with 10 μM ML 141, a recently characterizedinhibitor with selectivity for the active site of human CDC42, or withthe vehicle control polyethylene glycol (PEG, 18-20 h) followed byincubation with an invasive strain of S. aureus (ATCC 29213). ML 141decreased invasion at 1 h (FIGS. 4A-1 and 4A-2). Inhibition wasdetectable in all cell types examined (HEK 293A, U-87 MG, RAW 264.7, andA549; data not shown). To examine whether the intracellular bacterialpopulation within ML 141 treated host cells returned to control levelsover time, intracellular bacteria were examined 48 h post-invasion. Thenumber of viable bacteria recovered at 48 h was lower in ML 141 treatedcells compared to vehicle control (FIGS. 7 and 11).

Small Molecule Inhibition of CDC42 Limits Damage to Host Cells.

Invasion by S. aureus is progressively damaging to the host cell. Toexamine whether inhibition of invasion and sustained suppression of theintracellular bacterial population limits this damage, host cells weretreated with ML 141 or with vehicle control and examined at theultrastructural level 24 h post-invasion. Following infection,organelles that remained intact within ML 141 treated cells were notdetectable within vehicle control treated cells (FIG. 9). At the hostcell membrane, filopodia that form in response to infection were notdetected in ML 141 treated cells, yet were readily observed in thevehicle control. Bacteria within ML 141 treated cells were locatedwithin membranous boundaries. In vehicle control treated cells, bacteriawere cytosolic and appeared to be undergoing cellular division.Consistent with the invasion assays, fewer bacteria were observed withinML 141 treated cells.

Small Molecule Inhibition of CDC42 Decreases the Disassembly of ActinStructures During Infection.

In previous work, it was found that during invasion of HUVEC by S.aureus, actin stress fibers are disassembled. Here, it was found that ML141 treatment decreased the disassembly of actin stress fibers duringinfection (FIG. 8A).

Small Molecule Inhibition of CDC42 Decreases Adhesion Complex Formationand Function.

S. aureus uses adhesion complexes on the host cell membrane to gain hostcell entry. These complexes commonly are comprised of cytoskeletalproteins, including actin, tensin, and vinculin, and are organizedaround the central integrin receptor α5β1. S. aureus gains entry atthese complexes by attaching to host fibronectin, a ligand of α5β1. Asfibronectin binds to the α5β1 receptor and stimulates endocytic uptake,the attached bacteria are taken into the host cell as part of abacterial/fibronectin/receptor complex. It was previously found that theinhibition of invasion by simvastatin is associated with a decrease inthe abundance of these complexes. Here, ML 141 was used to explorewhether the decrease is due in part to loss of functional CDC42. ML 141treatment decreased adhesion complexes from 64% in vehicle controltreated cells to 37% in ML 141 treated cells (p≦0.001 by χ², FIG. 10A).The decrease in abundance might also be due to decreased expression ofthe central organizing integrin, 31. However, no difference in theexpression of 31 was detected between treatment groups (FIG. 10B).

Small Molecule Inhibition of CDC42 Decreases Adherence to Fibronectin.

The inhibition of invasion might be due in part to diminished adherenceto fibronectin, the ligand of α5β1 used by S. aureus for gaining hostcell entry. ML 141 treatment decreased adherence of HUVEC tofibronectin-coated plates from 24±4% to 14±2% (p<0.05 by Student'st-test, FIG. 10C).

Structural Analogs of ML 141 Limit Host Cell Invasion.

To improve the in vivo potency of ML 141, structural analogs weresynthesized. The analogs, designated as the RSM series (structures areavailable in Table 3), were assessed using the invasion assay.Intracellular bacterial levels were diminished by the analogs in the RSMseries (Tables 1 and 2). To verify that the apparent inhibition ofinvasion was not due to host cell death, cytotoxicity was assessed. RSM04 and RSM 26 were found to be cytotoxic (Table 4). Cytotoxicity was notdetected in response to ML 141 or in response to any additionalstructural analog (Tables 4 and 5). ML 141 was synthesized originallywithin a series of potential antimicrobials. To verify that the apparentinhibition of invasion was not due to a loss in numbers of viablebacteria, bactericidal activity was assessed. RSM 26 was found to bebactericidal (Table 6). Bactericidal activity was not detected inresponse to ML 141 or in response to any additional structural analog(Tables 6 and 7).

Experimental Section 5: ML 141 Inhibition of Cellular Invasion byStreptococcus Pyogenes Rationale

ML 141 may inhibit invasion by additional clinically importantintracellular pathogens, such as S. pyogenes, that use fibronectinbinding to gain host cell entry. For this, we examined an M+ strain ofStreptococcus pyogenes (90-226). The M protein is a fibronectin-bindingprotein of S. pyogenes that facilitates invasion.

Materials and Methods

Endothelial Cell Cultures, Bacterial Strains, and Growth Conditions:

Human umbilical vein endothelial cells (HUVEC, EMD Millipore, Billerica,Mass., SCCE001) were cultured in EndoGRO-LS complete media (EMDMillipore, SCME001) and maintained in 75 cm² vented cap flasks(Thermo-Fisher Scientific, Pittsburgh, Pa., 10-126-37) at 37° C./5% CO₂. Streptococcus pyogenes (90-226, M1+) was cultured in Todd Hewitt broth(THB, Thermo-Fisher Scientific, DF0492-17-6) and grown at 37° C. with noshaking.

Invasion Assay:

Two days prior to infection, S. pyogenes were cultured in 5 ml THB 18-24h from a single colony grown on THB/blood (Cleveland Scientific, Bath,Ohio, SBCIT-100) agar (Moorhead & Company, Rocklin, Calif., gelidium),and 10 μl subcultured 18 h prior to infection. HUVEC were seeded in24-well plates (Sigma-Aldrich, St. Louis, Mo., CLS3524) that had beencoated with Attachment Factor (Life Technologies, Carlsbad, Calif.,S006100) for 30 min at 37° C./5% CO₂. HUVEC were plated at a density of1.5×10⁵ cells/well. For infection, S. pyogenes were harvested bycentrifugation, 37° C./3 min/10,000 RPM, washed, and resuspended infresh 0.85% saline (NaCl, Thermo-Fisher Scientific, S-271-500). Bacteriawere diluted to 4.8×10⁸ colony forming units (CFU)/ml and incubated at37° C./5% CO₂ with HUVEC at multiplicity of infection and for durationindicated in each figure or table. CFU of 90-226 S. pyogenes had beendetermined from a growth curve, fitting an OD600 of 0.5 to 4.8×10⁸CFU/ml. After infection, each well was washed three times with sterile1× phosphate buffered saline (PBS, Life Technologies, 20012043) toremove unattached S. pyogenes. HUVEC were then incubated at 37° C., 5%CO₂ for 45 min in 10% fetal bovine serum (FBS, Atlanta Biologicals,Flowery Branch, Ga., S111S0)/PBS containing 500 μg/ml gentamicin(Sigma-Aldrich, G1272). Following the incubation with gentamicin toremove extracellular bacteria, HUVEC were washed three times with PBS.Sterile distilled ice-cold water (Life Technologies, 15230-162) wasadded to each well for 5 min at room temperature (RT) to permeabilizethe host cells. Serial dilutions of this bacteria-containing water wereperformed in saline, plated on THB/blood agar, and incubated for 18-20 hat 37° C. The following day, colonies were counted to determine CFU/mlrecovered from invasion.

Immunofluorescence:

For actin assessment, HUVEC were seeded at 6×10⁵ cells/ml in 35 mmglass-bottom dishes (MatTek, Ashland, Mass.) that had been coated withAttachment Factor. HUVEC were pretreated with ML 141 or with DMSO andtreated with gentamicin as described above. HUVEC were washed threetimes with PBS and fixed with 4% paraformaldehyde for 30 min, RT. HUVECwere gently washed four times with PBS, permeabilized and blocked with1% BSA/0.1% Triton (Sigma-Aldrich, T8787). HUVEC were gently washed fourtimes with PBS, probed for actin with Alexa Fluor 488 phalloidin (1:40,Life Technologies, A12379) in PBS (30 min, RT) and washed three moretimes with PBS. Actin stress fiber assessment was conducted using aZeiss Axiovert200 microscope equipped with a plan-apochromat 40×/1.2numerical aperture water immersion lens. A total of 200 HUVEC wereassessed for actin stress fiber disassembly from randomly selectedfields-of-view. Images were collected using a LSM 5 Pascal scan head.

Results

In response to ML 141 pretreatment (18-20 hours), intracellularbacterial populations were diminished following 2 hours of infection ata MOI of 30 (Table 8). When ML 141 was added at the time of infection atthe same MOI of 30, intracellular bacterial populations were notdiminished. However, when the MOI was reduced to 0.25, intracellularbacterial populations were lower in the ML 141 treatment group comparedto the vehicle control treatment group. Intracellular bacterialpopulations were lower 24 hours post-infection in the ML 141 treatmentgroup, indicating that the decrease was sustained over time. In order todetermine whether ML 141 could limit the progression of infection ifadministered after the onset of infection, ML 141 was added 1 hour afterinfection at this same MOI of 0.25, but no reduction of intracellularbacterial populations was detected. However, when the MOI was reduced to0.0025, intracellular bacterial populations were lower in the ML 141treatment group compared to the vehicle control treatment group 24 hourspost-infection. Taken together, these data suggest that ML 141 inhibitsinvasion by newly dividing, extracellular bacteria over time.

Similar to the inhibition of S. aureus invasion, inhibition of S.pyogenes invasion by ML 141 was reversible (FIG. 12) and was associatedwith less reordering of actin stress fibers (FIG. 13).

TABLE 8 Assessment of effect of ML 141 on Streptococcus pyogenesinvasion. HUVEC were treated with ML 141 (10 μM) or with dimethylsulfoxide as the vehicle control and incubated with Streptococcuspyogenes at the MOI indicated. Extracellular bacteria were removed bygentamicin, an antimicrobial with limited mammalian membranepermeability. Intracellular bacteria were released from HUVEC into themedium by incubation in cold water (5 min). Serial dilutions wereincubated (16 h) on Todd Hewitt broth/blood agar plates and coloniesenumerated to determine colony forming units (CFU)/ml. Data are %inhibition relative to control ± SEM (*P < 0.05 by Student's t-test; n =3-5/treatment). Duration Compound Addition of Infection MOI % Inhibition(± SEM) 18-20 hours pre- 2 h 30 96 ± 4%* infection Time of infection 2 h30 No difference Time of infection 2 h 0.25 81 ± 7%* Time of infection 2h 0.25  62 ± 10%* 1 hour post-infection 2 h 0.25 No difference 1 hourpost-infection 2 h 0.0025 92 ± 6%*

Pre-treatment of host cells with ML 141 or its analogs could limitinfection by clinically important pathogens reliant onfibronectin-binding for invasion by inhibition of CDC42. Suchfibronectin-reliant intracellular pathogens include Coxiella burnetii,Chlamydia, Legionella, and Bartonella, which use fibronectin-binding toinvade endocardial tissue during infective endocarditis, Mycobacteriumleprae, a causative agent of leprosy, and by Neisseria gonorrhoeae, thecausative agent of gonorrhea.

While the novel technology has been illustrated and described in detailin the figures and foregoing description, the same is to be consideredas illustrative and not restrictive in character, it being understoodthat only the preferred embodiments have been shown and described andthat all changes and modifications that come within the spirit of thenovel technology are desired to be protected. As well, while the noveltechnology was illustrated using specific examples, theoreticalarguments, accounts, and illustrations, these illustrations and theaccompanying discussion should by no means be interpreted as limitingthe technology. All patents, patent applications, and references totexts, scientific treatises, publications, and the like referenced inthis application are incorporated herein by reference in their entirety.

While this disclosure has been described as having an exemplary design,the present disclosure may be further modified within the spirit andscope of this disclosure. This application is therefore intended tocover any variations, uses, or adaptations of the disclosure using itsgeneral principles. Further, this application is intended to cover suchdepartures from the present disclosure as come within known or customarypractice in the art to which this disclosure pertains.

1. A method of suppressing bacterial infection comprising: administeringML 141 or its analogs to cells infected by Staphylococcus, where theanalogs are defined by the following structures:

where Ar is phenyl, acetamidophenyl (-Ph-NH—CO-Me), or propanamidophenyl(Ph-NH—CO-Et), R is hydrogen, R′ is hydrogen, and Ar′ is methoxyphenyl(-Ph-O-Me), acetamidophenyl (-Ph-NH—CO-Me), or dimethylaminophenyl(Ph-N-Me₂); and

where Ar is phenyl and Ar′ is methoxyphenyl (-Ph-O-Me).
 2. The method ofclaim 1 wherein said bacterial infection is from Staphylococcus aureus.3. The method of claim 1 wherein administering includes providing ML 141or its analogs adjacent to the cells.
 4. The method of claim 1 whereinadministering includes testing ML 141 on the cells.
 5. The method ofclaim 1 wherein administering includes testing at least one analog onthe cells.
 6. The method of claim 1 wherein suppressing bacterialinfection includes suppressing initial bacterial infection.
 7. Themethod of claim 1 wherein suppressing bacterial infection includessuppressing persistent bacterial infection.
 8. The method of claim 7wherein administering includes providing approximately 1 μM of ML 141 orits analogs.
 9. The method of claim 7 wherein administering includesproviding approximately 10 μM of ML 141 or its analogs.
 10. The methodof claim 1 wherein administering occurs subsequent to the onset ofbacterial infection.
 11. The method of claim 1 wherein the cells areanimal cells.
 12. The method of claim 1 wherein the cells are humancells.
 13. The method of claim 1 wherein the cells express CDC42.
 14. Amethod of suppressing bacterial infection, comprising: providing achemical defined by the following structures:

where Ar is phenyl, acetamidophenyl (-Ph-NH—CO-Me), or propanamidophenyl(Ph-NH—CO-Et), R is hydrogen, R′ is hydrogen, and Ar′ is methoxyphenyl(-Ph-O-Me), acetamidophenyl (-Ph-NH—CO-Me), or dimethylaminophenyl(Ph-N-Me₂); and

where Ar is phenyl and Ar′ is methoxyphenyl (-Ph-O-Me); and providingthe chemical to cells infected by Staphylococcus.
 15. The method ofclaim 14 further comprising providing a pharmaceutically acceptedsolvent or delivery vehicle selected from the group consisting ofpolyethylene glycol (PEG), dimethyl sulfoxide, ethanol, and combinationsthereof.
 16. The method of claim 15 wherein the solvent or deliveryvehicle is polyethylene glycol.
 17. A method of suppressing bacterialinfection comprising: administering ML 141 or its analogs to a patientinfected by bacteria of the genus Staphylococcus, where the analogs aredefined by the following structures:

where Ar is phenyl, acetamidophenyl (-Ph-NH—CO-Me), or propanamidophenyl(Ph-NH—CO-Et), R is hydrogen, R′ is hydrogen, and Ar′ is methoxyphenyl(-Ph-O-Me), acetamidophenyl (-Ph-NH—CO-Me), or dimethylaminophenyl(Ph-N-Me₂); and

where Ar is phenyl and Ar′ is methoxyphenyl (-Ph-O-Me).